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Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae)
Zoology 114 (2011) 298–305
Contents lists available at ScienceDirect
Zoology journal homepage: www.elsevier.de/zool
Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae) Lilian Franco-Belussi ∗ , Classius de Oliveira Department of Biology, São Paulo State University (UNESP), São José do Rio Preto, 15054-000 São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 1 March 2011 Received in revised form 2 April 2011 Accepted 8 May 2011 Keywords: Amphibians Endotoxemia Melanin Pigment cells
a b s t r a c t Amphibians have an extracutaneous pigmentary system composed of melanin-containing cells in various tissues and organs. The functional role of these pigment cells in visceral organs has not yet been determined, although several hypotheses have been proposed. Our aim was to describe the visceral pigmentation in the frog Eupemphix nattereri under conditions of endotoxemia induced experimentally with lipopolysaccharides (LPS) from Escherichia coli and to analyze the pigmentation on the organs’ surface. We used 60 adult males of E. nattereri and analyzed the visceral pigmentation 2 (LPS 2 h), 6 (LPS 6 h), 12 (LPS 12 h), 24 (LPS 24 h) or 48 h (LPS 48 h) after the LPS inoculation. We observed pigmentation on the surface of several abdominal organs. The highest degree of pigmentation was found only on the testes of the animals in the LPS 2 h, LPS 6 h and LPS 12 h groups. The pigmentation decreased in the animals of the LPS 24 h and LPS 48 h groups. The LPS administration produced no changes in the pigmentation of the cardio-respiratory and digestive systems. Thus, the cells appear to have responded to LPS intoxication, producing a rapid increase of pigmentation on the surface of the testes and a subsequent decrease in the pigmentation. These changes are most likely related to the bactericidal role of melanin, which neutralizes the effects of LPS. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Amphibians have an extracutaneous pigmentary system composed of melanin-containing cells in various tissues and organs (Gallone et al., 2002), such as the liver, spleen (Agius, 1980; Agius and Agbede, 1984; Zuasti et al., 1990; Christiansen et al., 1996; Gallone et al., 2002), kidneys (Agius, 1980; Zuasti et al., 1989), parietal peritoneum (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), lung (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), skeletal muscles (Divya et al., 2010), heart (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), blood vessels (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), testes (Zieri et al., 2007; Franco-Belussi et al., 2009; Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), mesentery (Zuasti et al., 1990) and meninges (Zuasti et al., 1998; Bagnara and Matsumoto, 2006). The pigment cells from the epidermis and various organs are similar to melanocytes (Agius and Agbede, 1984; Zuasti et al., 1998) derived from the ectodermal neural crest (Sichel et al., 1997). These melanocytes occur in organs and are often located in the connective tissue (capsule and interstitium) or are externally associated with the tunica adventitia or serosa (Gallone et al., 2002; FrancoBelussi et al., 2011). The pigment cells in hematopoietic organs also
∗ Corresponding author. Tel.: +55 17 3221 2387; fax: +55 17 3221 2390. E-mail address: [email protected] (L. Franco-Belussi). 0944-2006/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2011.05.001
have phagocytic activity, similar to that of macrophages. These cells produce and store melanin, which absorbs and neutralizes free radicals, cations and other potentially toxic agents derived from the degradation of phagocytized cellular material (Zuasti et al., 1989; Agius and Roberts, 2003). The functional role of these pigment cells in visceral organs has not yet been defined, although several hypotheses have been proposed (Gallone et al., 2002), including cytoprotective functions against free radicals (McGraw, 2005) and detoxification from pollutants (Fenoglio et al., 2005). Other cytoprotective functions include protection against bacteria in ectothermic vertebrates, mainly at low temperatures (Christiansen et al., 1996). This protection is very important in these animals because they are commonly in contact with Escherichia coli in the habitats where they live. E. coli is a Gram-negative bacterium whose pathogenicity is related to the lipopolysaccharides (LPS) present in its cell wall. This pathogenic agent is an endotoxin that triggers inflammatory responses in the host organism, stimulating mononuclear phagocytes to produce cytokines (Flores Quintana and Ruas de Moraes, 2001). Since melanin has a protective role against bacterial effects, we would expect that the visceral pigmentation will be increased after infection. Therefore, our aims in this study are to describe the visceral pigmentation in Eupemphix nattereri treated with lipopolysaccharides (LPS) from E. coli and to test whether the LPS alter the superficial pigmentation of the organs in this species.
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Fig. 1. Organs and structures of the abdominal cavity of Eupemphix nattereri. (A and B) Cardio-respiratory system. (C and D) Organs and structures of the digestive system. Abbreviations: Gb, gallbladder; H, heart; I, intestine; M, mesentery; L, lung; S, stomach.
2. Materials and methods We used 60 adult males of E. nattereri (Anura: Leiuperidae) collected from permanent and temporary ponds during the breeding season (January 2011) in the vicinity of São José do Rio Preto, São Paulo State, Brazil. The handling of animals and all experimental procedures were approved by and followed the recommendations of the Committee on Ethics and Animal Experimentation of São Paulo State University (UNESP) (Protocol # 038/2011-CEUA). The specimens captured were placed in plastic bags, transported to the laboratory and housed in terraria (28 cm × 21 cm × 15 cm) with 5 cm of soil at the bottom; they were provided with food and water ad libitum. The animals remained in acclimatization at room temperature for 7 days prior to the experiments. After the experimental treatment, the animals were transferred to sterilized terraria of the same size as the previous ones but without soil. This procedure prevented secondary infections during the experiments. We administered a single intraperitoneal dose of LPS (3 mg/kg) from E. coli (Serotype 0127:B8; Sigma–Aldrich, St. Louis, MO, USA) diluted in sterile saline solution (adapted from Flores Quintana and Ruas de Moraes, 2001) to each of 50 animals. We analyzed 10 animals 2 h after inoculation (LPS 2 h group, hereafter), 10 animals 6 h after inoculation (LPS 6 h group, hereafter), 10 animals 12 h after inoculation (LPS 12 h group, hereafter), 10 animals 24 h after
inoculation (LPS 24 h group, hereafter) and 10 animals 48 h after inoculation (LPS 48 h group, hereafter). These examination times were based on information in Flores Quintana and Ruas de Moraes (2001). These authors demonstrated an increase of pigment cells in the kidneys of fish after 24 h. Therefore, we adopted the experimental times of 2, 6 and 12 h to characterize the immediate response to LPS and the experimental times of 24 and 48 h to characterize the late response to LPS. Ten animals each received an injection of sterile saline to serve as a control group. We anesthetized and euthanized the animals with a lethal dose of benzocaine diluted in water (1.0 g/l). The dissection was made through a median incision from the cloaca to the pectoral girdle. The photo documentation was made with a stereoscopic microscope (Leica MZ16; Leica Microsystems GmbH, Wetzlar, Germany) connected to an image-capturing system. We used the protocol proposed by Franco-Belussi et al. (2009) to describe the structural and qualitative features of pigment cells associated with visceral organs and tissues. This protocol was developed to quantify the intensity of pigmentation on the testes of anurans, and it was applied to other organs and tissues in this study. The classification is based on the intensity of pigmentation, ranging from absence (category 0), in which pigmented cells are completely absent from the surface of the organs, to an intense dark pigmentation (category 3), in which a massive amount of pigmented cells
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Fig. 2. Organs and structures of the abdominal cavity of Eupemphix nattereri. (A) Final portion of the digestive system (rectum). (B and C) Urogenital system. (D) Other regions in which pigment cells were found. Abbreviations: K, kidney; Ls, lumbosacral parietal peritoneum; Pl, nerves of the lumbar plexus; R, rectum; Sl, spleen; T, testis; Ub, urinary bladder; V, vertebral column.
produces an intensely pigmented structure and an evident alteration of the usual color of the organ. Category 1 is characterized by a slight pigmentation with a few pigmented cells, and category 2 by a large quantity of pigmented cells.
In this study, we applied this protocol to 12 anatomical regions: (1) the pericardium and cardiac blood vessels; (2) the heart; (3) the lungs; (4) the stomach; (5) the intestine; (6) the rectum; (7) the kidneys and renal blood vessels; (8) the testicles; (9) the urinary
Fig. 3. Variation in the pigmentation on the testes of Eupemphix nattereri. (A) Presence of a few pigmented cells (category 1). (B) Moderate pigmentation: the normal color of the organ is not visible (category 2). (C) Intense pigmentation (category 3).
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Fig. 4. Comparative analysis of the pigmentation of organs and structures of the cardio-respiratory system in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
Fig. 5. Differences in the categories of pigmentation on the heart, kidneys and testes of all experimental groups. Mean ± standard error. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively. Different letters represent statistical differences (P ≤ 0.05) among different experimental groups for the same organ.
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Fig. 6. Comparative analysis of the pigmentation of organs and structures of the digestive system in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
bladder; (10) the nerves of the lumbar plexus; (11) the lumbosacral parietal peritoneum; and (12) the mesentery. To compare the differences among categories of pigmentation in each organ or region, we used the G test for goodness of fit, with Yates’ correction (Sokal and Rohlf, 1995). This test was implemented using the code provided by Peter Hurd (available at http://www.psych.ualberta.ca/∼phurd/cruft/g.test.r). The test was run using the software R v. 2.11.1 (R Development Core Team, 2010). 3. Results We observed conspicuous pigmentation on the surface of several abdominal organs (Figs. 1–3). The heart (cardiac muscle) had no pigmentation in the control and LPS 24 h groups, whereas the heart of the animals in the LPS 2 h, LPS 6 h, LPS 12 h and LPS 48 h groups had slight pigmentation. We observed an increase in the pigmentation of the surface of the heart in the experimental animals of the LPS 12 h and LPS 48 h groups (G = 62.73; df = 15; p < 0.0001; Figs. 4 and 5). The lungs, pericardium, and cardiac blood vessels were also pigmented to some degree (categories 1 and 2) in all the individuals (Figs. 1 and 4). There were no significant variations among the experimental groups in the amount of pigmentation on the pericardium and cardiac blood vessels (G = 7.31; df = 15; p = 0.95). Slight pigmentation (category 1) was frequent in all the animals. The majority of the individuals in the LPS 2 h group had a significant amount of pigmentation (category 2) on the lungs. In the
control and other experimental groups, slight pigmentation (category 1) occurred frequently in this organ, with some individuals of the control and the LPS 2 h, LPS 6 h and LPS 24 h groups showing category 2 of pigmentation. However, these differences in the amount of pigmentation were not significant (G = 15.77; df = 15; p = 0.40). No differences were found between the lung antimeres (data not shown). The organs of the digestive system (Figs. 1C,D and 2A) showed little pigmentation on their surfaces. The stomach and intestine of all the animals examined had no pigmentation, with the exception of the animals of the LPS 24 h group, which had slight pigmentation (category 1) on the intestine. However, this variation was not significant (G = 7.53; df = 15; p = 0.94). The majority of the individuals exhibited a slight amount of pigmentation (category 1) on the rectum. The individuals of the control and LPS 24 h groups had a high amount of pigmentation (category 2) in this region (Fig. 6), but this variation was not significant (G = 9.38; df = 15; p = 0.86). We observed no pigmentation on the urinary bladder in any of the animals (Fig. 2B). Slight to moderate pigmentation (categories 1 and 2) was found on the dorsal surface of the kidneys and the renal blood vessels of all the animals (Figs. 2C and 7). The groups LPS 6 h and LPS 12 h showed an increase in the pigmentation on the surfaces of these organs (G = 42.00; df = 15; p < 0.0001; Fig. 5). The organ in which we found the greatest effect of LPS was the testis. The amount of pigmentation on the testes of the animals of the LPS 2 h, LPS 6 h and LPS 12 h groups (G = 47.07; df = 15; p < 0.0001) increased relative to the control group. A decrease in the amount
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Fig. 7. Comparative analysis of the pigmentation of organs and structures of the urogenital system in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
of pigmentation on the testes, relative to the amounts seen in the other experimental groups, was found in the animals of the LPS 24 h and LPS 48 h groups (Figs. 3, 5 and 7). No differences in pigmentation were found between the gonadal and renal antimeres. The pigmentation on the mesentery, the nerves of the lumbar plexus and the lumbosacral parietal peritoneum (Fig. 2D) ranged from low to moderate (categories 1 and 2) in all experimental groups (mesentery: G = 31.68; df = 15; p = 0.007; lumbosacral parietal peritoneum: G = 8.25; df = 15; p = 0.91; and lumbar nerve plexus: G = 12.91; df = 15; p = 0.61). These results were not related to the effects of LPS (Fig. 8).
4. Discussion The pigmentation on the surface of the testes increased rapidly (approximately 2 h after inoculation) in response to the exposure to LPS and decreased to control levels 24 h after systemic administration of LPS. This immediate increase was also evident on the kidneys, where we observed an increase of pigmentation 6 h after LPS exposure. In contrast, the pigmentation on the heart increased only after 12 h. However, the pigmentation did not vary in other cardiorespiratory organs. No changes related to LPS administration were observed in the organs of the digestive system or in the mesentery, the nerves of the lumbar plexus and the lumbosacral parietal peritoneum.
The function of the pigmentation in the visceral organs of frogs is not yet clear. Melanin has a protective role against free radicals and other potentially toxic agents (Zuasti et al., 1989; Agius and Roberts, 2003) and is a bactericidal agent (Christiansen et al., 1996). We observed an increase in the pigmentation of some organs after LPS exposure. Pigmentation increased on the testes. The protective role of melanin against the toxic effects of endotoxemia (i.e., oxygen burst and oxidative and nitrosative stress) may be involved in this response. Testicular dysfunction after contact with LPS has been reported in mammals: the germ cells undergo apoptosis and spermatogenesis is affected (Kajihara et al., 2006; Reddy et al., 2006). In the current study, 24 h after LPS administration, the pigmentation of the testes returned to control levels. Agius and Roberts (2003) reported an increase in pigmentation in the hematopoietic organs of fish related to pathology. This finding suggests that these cells and the pigmented cells present on the testes, the kidneys and the heart may have a similar functional role. Divya et al. (2010) reported a signaling action of melanocytes in the tail skeletal muscle of tadpoles at metamorphosis. This result suggests that melanin plays a role in the cascade of events leading to tail resorption in anuran larvae. Pigment cells on the testes are not described for most species, although some authors (e.g., Oliveira et al., 2002, 2003; Oliveira and Zieri, 2005; Zieri et al., 2007) have found a close relationship between visceral melanocytes and the vascular system as well as with the blood vessels and the associated connective membranes of
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Fig. 8. Comparative analysis of the pigmentation of the mesentery, parietal peritoneum and lumbar nerve plexus in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
other organs. The pigmentation is present in the interstitium and tunica albuginea of the testes of E. nattereri, Physalaemus cuvieri and Physalaemus marmoratus and gives the testes a completely or partially black appearance (Oliveira et al., 2002, 2003; Oliveira and Zieri, 2005; Zieri et al., 2007). The association of pigmentation with the germinative locules suggests that pigmented cells may be protecting germ cells against free radicals. The visceral pigmentation of internal organs varies depending on intrinsic and extrinsic factors. The intrinsic factors include age and the nutritional and pathological status of the animal (Agius and Agbede, 1984). In addition, low temperatures have been shown to increase the amount of melanin and to induce metabolic and structural changes in hepatic cells of Rana esculenta (Barni et al., 1999).
The visceral pigmentation on the testes of Rhinella schneideri changes during the reproductive period. The pigmentation on the surface of these organs increases at the end of the reproductive season (Moresco and Oliveira, 2009). This observation is consistent with the action of melanin during spermatogenesis, a period with high testicular activity. Nevertheless, in other regions, such as the pericardium, the heart, the stomach, the intestine, the rectum, the nerves of the lumbar plexus, the lumbosacral parietal peritoneum and the mesentery, there were no changes in surface pigmentation related to the reproductive period (Moresco and Oliveira, 2009). However, the systemic administration of LPS promoted an immediate increase of pigmentation in the kidneys and the heart. This result demonstrates that visceral cells react against an actual product of bacteremia by producing melanin.
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The LPS from E. coli administered experimentally caused changes in the superficial pigmentation of the testes, kidney and heart. This observation suggests that this visceral pigmentation is a response to one or more products of LPS exposure. Therefore, these findings could be explained by the bactericidal role of melanin (Christiansen et al., 1996) because melanin protects cells and organs against products of LPS exposure. In the testes, the pigmented cells protect the germ cells against products of real bacteremia that could cause damage and influence the reproductive output directly, thus decreasing the fitness of the animals. Acknowledgements We thank Diogo B. Provete, M.Sc., Rafaela M. Moresco, M.Sc., and Dr. Amilcar S. Damazo for suggestions on earlier drafts of the manuscript, Dr. Rodrigo Zieri and Dr. Lia R. de Souza Santos for helping with field work and the Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for providing grants (05/02919-5 and 2009/13925-7) and awarding a master’s scholarship to FLB (Proc. 2008/52389-0). We also thank IBAMA for the collecting permits (RAN-IBAMA 18573-1). References Agius, C., 1980. Phylogenetic development of melano-macrophage centers in fish. J. Zool. 191, 11–31. Agius, C., Agbede, S.A., 1984. An electron microscopical study on the genesis of lipofuscin, melanin and haemosiderin in the haemopoietic tissues of fish. J. Fish Biol. 24, 471–488. Agius, C., Roberts, R.J., 2003. Review: melano-macrophage centres and their role in fish pathology. J. Fish Biol. 26, 499–509. Bagnara, J., Matsumoto, J., 2006. Comparative anatomy and physiology of pigment cells in non-mammalian tissues. In: Nordlund, J.J., Boissy, R.E., Hearing, V.J., King, R.A., Ortonne, J.P. (Eds.), The Pigmentary System: Physiology and Pathophysiology. Oxford University Press, New York, pp. 9–40. Barni, S., Bertone, V., Croce, A.C., Bottiroli, G., Bernini, F., Gerzeli, G., 1999. Increase in liver pigmentation during natural hibernation in some amphibians. J. Anat. 195, 19–25. Christiansen, J.L., Grzybowski, J.M., Kodama, R.M., 1996. Melanomacrophage aggregations and their age relationships in the yellow mud turtle, Kinosternon flavescens (Kinosternidae). Pigm. Cell Res. 9, 185–190. Divya, L., Beyo, R.S., Sreejith, P., Akbarsha, M.A., Oommen, V.O., 2010. Skeletal muscle–melanocyte association during tadpole tail resorption in a tropical frog, Clinotarsus curtipes Jerdon (Anura, Ranoidea). Zoology 113, 175–183. Fenoglio, C., Boncompagni, E., Fasola, M., Gandini, C., Comizzoli, S., Milanesi, G., Barni, S., 2005. Effects of environmental pollution on the liver parenchymal cells and
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Kupffer-melanomacrophagic cells of the frog Rana esculenta. Ecotoxicol. Environ. Saf. 60, 259–268. Flores Quintana, C., Ruas de Moraes, F., 2001. Respuesta inflamatoria a la inoculación de LPS en Pacu (Piaractus mesopotamicus) suplementados con cromo. Rev. Ictiol. 9, 13–19. Franco-Belussi, L., Zieri, R., Santos, L.R.S., Moresco, R.M., Oliveira, C., 2009. Pigmentation in anuran testes: anatomical pattern and variation. Anat. Rec. 292, 178–182. Franco-Belussi, L., Santos, L.R.S., Zieri, R., Oliveira, C., 2011. Visceral pigmentation in Dendropsophus (Anura: Hylidae): occurrence and comparison. Zool. Anz. 250, 102–110. Gallone, A., Guida, G., Maida, I., Cicero, R., 2002. Spleen and liver pigmented macrophages of Rana esculenta L. A new melanogenic system? Pigm. Cell Res. 15, 32–40. Kajihara, T., Okagaki, R., Ishihara, O., 2006. LPS-induced transient testicular dysfunction accompanied by apoptosis of testicular germ cells in mice. Med. Mol. Morphol. 39, 203–208. McGraw, K.J., 2005. The antioxidant function of many animal pigments: are there consistent health benefits of sexually selected colourants? Rev. Anim. Behav. 69, 757–764. Moresco, R.M., Oliveira, C., 2009. A comparative study of the extracutaneous pigmentary system in three anuran amphibian species evaluated during the breeding season. S. Am. J. Herpetol. 4, 1–8. Oliveira, C., Zieri, R., 2005. Pigmentac¸ão testicular em Physalaemus nattereri (Steindachner) (Amphibia, Anura) com observac¸ões anatômicas sobre o sistema pigmentar extracutâneo. Rev. Bras. Zool. 22, 454–460. Oliveira, C., Zanetoni, C., Zieri, R., 2002. Morphological observations on the testes of Physalaemus curvieri (Amphibia, Anura). Rev. Chil. Anat. 20, 263–268. Oliveira, C., Sant’ana, A.C., Omena, P.M., Santos, L.R.S., Zieri, R., 2003. Morphological considerations on the seminiferous structures and testes of anuran amphibians: Bufo crucifer, Physalaemus curvieri and Scinax fuscovarius. Biociências 11, 39–46. R Development Core Team, 2010. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. Available at: http://www.R-project.org (accessed on 20 June 2010). Reddy, M., Mahipal, S., Subhashini, J., Reddy, M.C., Roy, K.R., Reddy, G.V., Reddy, P.R.K., Reddanna, P., 2006. Bacterial lipopolysaccharide-induced oxidative stress in the impairment of steroidogenesis and spermatogenesis in rats. Rep. Toxicol. 22, 493–500. Sichel, G., Scalia, M., Mondio, F., Corsaro, C., 1997. The amphibian Kupffer cells build and demolish melanosomes: an ultrastructural point of view. Pigm. Cell Res. 10, 271–287. Sokal, R.R., Rohlf, F.J., 1995. Biometry: The Principles and Practice of Statistics in Biological Research, 3rd ed. W.H. Freeman &Co., New York. Zieri, R., Taboga, S.R., Oliveira, C., 2007. Melanocytes in the testes of Eupemphix nattereri (Anura, Leiuperidae): histological, estereological and ultrastructural aspects. Anat. Rec. 290, 795–800. Zuasti, A., Jara, J.R., Ferrer, C., Solano, F., 1989. Occurrence of melanin granules and melanosynthesis in the kidney of Sparus auratus. Pigm. Cell Res. 2, 93–100. Zuasti, A., Ferrer, C., Aroca, P., Solano, F., 1990. Distribution of extracutaneous melanin pigment in Sparus auratus, Mugil cephalus, and Dicentrarchus labrax (Pisces, Teleostei). Pigm. Cell Res. 3, 126–131. Zuasti, A., Jiménez-Cervantes, C., Gargia-Borrón, J.C., Ferrer, C., 1998. The melanogenic system of Xenopus laevis. Arch. Histol. Cytol. 61, 305–316.
Contents lists available at ScienceDirect
Zoology journal homepage: www.elsevier.de/zool
Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae) Lilian Franco-Belussi ∗ , Classius de Oliveira Department of Biology, São Paulo State University (UNESP), São José do Rio Preto, 15054-000 São Paulo, Brazil
a r t i c l e
i n f o
Article history: Received 1 March 2011 Received in revised form 2 April 2011 Accepted 8 May 2011 Keywords: Amphibians Endotoxemia Melanin Pigment cells
a b s t r a c t Amphibians have an extracutaneous pigmentary system composed of melanin-containing cells in various tissues and organs. The functional role of these pigment cells in visceral organs has not yet been determined, although several hypotheses have been proposed. Our aim was to describe the visceral pigmentation in the frog Eupemphix nattereri under conditions of endotoxemia induced experimentally with lipopolysaccharides (LPS) from Escherichia coli and to analyze the pigmentation on the organs’ surface. We used 60 adult males of E. nattereri and analyzed the visceral pigmentation 2 (LPS 2 h), 6 (LPS 6 h), 12 (LPS 12 h), 24 (LPS 24 h) or 48 h (LPS 48 h) after the LPS inoculation. We observed pigmentation on the surface of several abdominal organs. The highest degree of pigmentation was found only on the testes of the animals in the LPS 2 h, LPS 6 h and LPS 12 h groups. The pigmentation decreased in the animals of the LPS 24 h and LPS 48 h groups. The LPS administration produced no changes in the pigmentation of the cardio-respiratory and digestive systems. Thus, the cells appear to have responded to LPS intoxication, producing a rapid increase of pigmentation on the surface of the testes and a subsequent decrease in the pigmentation. These changes are most likely related to the bactericidal role of melanin, which neutralizes the effects of LPS. © 2011 Elsevier GmbH. All rights reserved.
1. Introduction Amphibians have an extracutaneous pigmentary system composed of melanin-containing cells in various tissues and organs (Gallone et al., 2002), such as the liver, spleen (Agius, 1980; Agius and Agbede, 1984; Zuasti et al., 1990; Christiansen et al., 1996; Gallone et al., 2002), kidneys (Agius, 1980; Zuasti et al., 1989), parietal peritoneum (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), lung (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), skeletal muscles (Divya et al., 2010), heart (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), blood vessels (Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), testes (Zieri et al., 2007; Franco-Belussi et al., 2009; Moresco and Oliveira, 2009; Franco-Belussi et al., 2011), mesentery (Zuasti et al., 1990) and meninges (Zuasti et al., 1998; Bagnara and Matsumoto, 2006). The pigment cells from the epidermis and various organs are similar to melanocytes (Agius and Agbede, 1984; Zuasti et al., 1998) derived from the ectodermal neural crest (Sichel et al., 1997). These melanocytes occur in organs and are often located in the connective tissue (capsule and interstitium) or are externally associated with the tunica adventitia or serosa (Gallone et al., 2002; FrancoBelussi et al., 2011). The pigment cells in hematopoietic organs also
∗ Corresponding author. Tel.: +55 17 3221 2387; fax: +55 17 3221 2390. E-mail address: [email protected] (L. Franco-Belussi). 0944-2006/$ – see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.zool.2011.05.001
have phagocytic activity, similar to that of macrophages. These cells produce and store melanin, which absorbs and neutralizes free radicals, cations and other potentially toxic agents derived from the degradation of phagocytized cellular material (Zuasti et al., 1989; Agius and Roberts, 2003). The functional role of these pigment cells in visceral organs has not yet been defined, although several hypotheses have been proposed (Gallone et al., 2002), including cytoprotective functions against free radicals (McGraw, 2005) and detoxification from pollutants (Fenoglio et al., 2005). Other cytoprotective functions include protection against bacteria in ectothermic vertebrates, mainly at low temperatures (Christiansen et al., 1996). This protection is very important in these animals because they are commonly in contact with Escherichia coli in the habitats where they live. E. coli is a Gram-negative bacterium whose pathogenicity is related to the lipopolysaccharides (LPS) present in its cell wall. This pathogenic agent is an endotoxin that triggers inflammatory responses in the host organism, stimulating mononuclear phagocytes to produce cytokines (Flores Quintana and Ruas de Moraes, 2001). Since melanin has a protective role against bacterial effects, we would expect that the visceral pigmentation will be increased after infection. Therefore, our aims in this study are to describe the visceral pigmentation in Eupemphix nattereri treated with lipopolysaccharides (LPS) from E. coli and to test whether the LPS alter the superficial pigmentation of the organs in this species.
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Fig. 1. Organs and structures of the abdominal cavity of Eupemphix nattereri. (A and B) Cardio-respiratory system. (C and D) Organs and structures of the digestive system. Abbreviations: Gb, gallbladder; H, heart; I, intestine; M, mesentery; L, lung; S, stomach.
2. Materials and methods We used 60 adult males of E. nattereri (Anura: Leiuperidae) collected from permanent and temporary ponds during the breeding season (January 2011) in the vicinity of São José do Rio Preto, São Paulo State, Brazil. The handling of animals and all experimental procedures were approved by and followed the recommendations of the Committee on Ethics and Animal Experimentation of São Paulo State University (UNESP) (Protocol # 038/2011-CEUA). The specimens captured were placed in plastic bags, transported to the laboratory and housed in terraria (28 cm × 21 cm × 15 cm) with 5 cm of soil at the bottom; they were provided with food and water ad libitum. The animals remained in acclimatization at room temperature for 7 days prior to the experiments. After the experimental treatment, the animals were transferred to sterilized terraria of the same size as the previous ones but without soil. This procedure prevented secondary infections during the experiments. We administered a single intraperitoneal dose of LPS (3 mg/kg) from E. coli (Serotype 0127:B8; Sigma–Aldrich, St. Louis, MO, USA) diluted in sterile saline solution (adapted from Flores Quintana and Ruas de Moraes, 2001) to each of 50 animals. We analyzed 10 animals 2 h after inoculation (LPS 2 h group, hereafter), 10 animals 6 h after inoculation (LPS 6 h group, hereafter), 10 animals 12 h after inoculation (LPS 12 h group, hereafter), 10 animals 24 h after
inoculation (LPS 24 h group, hereafter) and 10 animals 48 h after inoculation (LPS 48 h group, hereafter). These examination times were based on information in Flores Quintana and Ruas de Moraes (2001). These authors demonstrated an increase of pigment cells in the kidneys of fish after 24 h. Therefore, we adopted the experimental times of 2, 6 and 12 h to characterize the immediate response to LPS and the experimental times of 24 and 48 h to characterize the late response to LPS. Ten animals each received an injection of sterile saline to serve as a control group. We anesthetized and euthanized the animals with a lethal dose of benzocaine diluted in water (1.0 g/l). The dissection was made through a median incision from the cloaca to the pectoral girdle. The photo documentation was made with a stereoscopic microscope (Leica MZ16; Leica Microsystems GmbH, Wetzlar, Germany) connected to an image-capturing system. We used the protocol proposed by Franco-Belussi et al. (2009) to describe the structural and qualitative features of pigment cells associated with visceral organs and tissues. This protocol was developed to quantify the intensity of pigmentation on the testes of anurans, and it was applied to other organs and tissues in this study. The classification is based on the intensity of pigmentation, ranging from absence (category 0), in which pigmented cells are completely absent from the surface of the organs, to an intense dark pigmentation (category 3), in which a massive amount of pigmented cells
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Fig. 2. Organs and structures of the abdominal cavity of Eupemphix nattereri. (A) Final portion of the digestive system (rectum). (B and C) Urogenital system. (D) Other regions in which pigment cells were found. Abbreviations: K, kidney; Ls, lumbosacral parietal peritoneum; Pl, nerves of the lumbar plexus; R, rectum; Sl, spleen; T, testis; Ub, urinary bladder; V, vertebral column.
produces an intensely pigmented structure and an evident alteration of the usual color of the organ. Category 1 is characterized by a slight pigmentation with a few pigmented cells, and category 2 by a large quantity of pigmented cells.
In this study, we applied this protocol to 12 anatomical regions: (1) the pericardium and cardiac blood vessels; (2) the heart; (3) the lungs; (4) the stomach; (5) the intestine; (6) the rectum; (7) the kidneys and renal blood vessels; (8) the testicles; (9) the urinary
Fig. 3. Variation in the pigmentation on the testes of Eupemphix nattereri. (A) Presence of a few pigmented cells (category 1). (B) Moderate pigmentation: the normal color of the organ is not visible (category 2). (C) Intense pigmentation (category 3).
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Fig. 4. Comparative analysis of the pigmentation of organs and structures of the cardio-respiratory system in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
Fig. 5. Differences in the categories of pigmentation on the heart, kidneys and testes of all experimental groups. Mean ± standard error. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively. Different letters represent statistical differences (P ≤ 0.05) among different experimental groups for the same organ.
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Fig. 6. Comparative analysis of the pigmentation of organs and structures of the digestive system in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
bladder; (10) the nerves of the lumbar plexus; (11) the lumbosacral parietal peritoneum; and (12) the mesentery. To compare the differences among categories of pigmentation in each organ or region, we used the G test for goodness of fit, with Yates’ correction (Sokal and Rohlf, 1995). This test was implemented using the code provided by Peter Hurd (available at http://www.psych.ualberta.ca/∼phurd/cruft/g.test.r). The test was run using the software R v. 2.11.1 (R Development Core Team, 2010). 3. Results We observed conspicuous pigmentation on the surface of several abdominal organs (Figs. 1–3). The heart (cardiac muscle) had no pigmentation in the control and LPS 24 h groups, whereas the heart of the animals in the LPS 2 h, LPS 6 h, LPS 12 h and LPS 48 h groups had slight pigmentation. We observed an increase in the pigmentation of the surface of the heart in the experimental animals of the LPS 12 h and LPS 48 h groups (G = 62.73; df = 15; p < 0.0001; Figs. 4 and 5). The lungs, pericardium, and cardiac blood vessels were also pigmented to some degree (categories 1 and 2) in all the individuals (Figs. 1 and 4). There were no significant variations among the experimental groups in the amount of pigmentation on the pericardium and cardiac blood vessels (G = 7.31; df = 15; p = 0.95). Slight pigmentation (category 1) was frequent in all the animals. The majority of the individuals in the LPS 2 h group had a significant amount of pigmentation (category 2) on the lungs. In the
control and other experimental groups, slight pigmentation (category 1) occurred frequently in this organ, with some individuals of the control and the LPS 2 h, LPS 6 h and LPS 24 h groups showing category 2 of pigmentation. However, these differences in the amount of pigmentation were not significant (G = 15.77; df = 15; p = 0.40). No differences were found between the lung antimeres (data not shown). The organs of the digestive system (Figs. 1C,D and 2A) showed little pigmentation on their surfaces. The stomach and intestine of all the animals examined had no pigmentation, with the exception of the animals of the LPS 24 h group, which had slight pigmentation (category 1) on the intestine. However, this variation was not significant (G = 7.53; df = 15; p = 0.94). The majority of the individuals exhibited a slight amount of pigmentation (category 1) on the rectum. The individuals of the control and LPS 24 h groups had a high amount of pigmentation (category 2) in this region (Fig. 6), but this variation was not significant (G = 9.38; df = 15; p = 0.86). We observed no pigmentation on the urinary bladder in any of the animals (Fig. 2B). Slight to moderate pigmentation (categories 1 and 2) was found on the dorsal surface of the kidneys and the renal blood vessels of all the animals (Figs. 2C and 7). The groups LPS 6 h and LPS 12 h showed an increase in the pigmentation on the surfaces of these organs (G = 42.00; df = 15; p < 0.0001; Fig. 5). The organ in which we found the greatest effect of LPS was the testis. The amount of pigmentation on the testes of the animals of the LPS 2 h, LPS 6 h and LPS 12 h groups (G = 47.07; df = 15; p < 0.0001) increased relative to the control group. A decrease in the amount
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Fig. 7. Comparative analysis of the pigmentation of organs and structures of the urogenital system in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
of pigmentation on the testes, relative to the amounts seen in the other experimental groups, was found in the animals of the LPS 24 h and LPS 48 h groups (Figs. 3, 5 and 7). No differences in pigmentation were found between the gonadal and renal antimeres. The pigmentation on the mesentery, the nerves of the lumbar plexus and the lumbosacral parietal peritoneum (Fig. 2D) ranged from low to moderate (categories 1 and 2) in all experimental groups (mesentery: G = 31.68; df = 15; p = 0.007; lumbosacral parietal peritoneum: G = 8.25; df = 15; p = 0.91; and lumbar nerve plexus: G = 12.91; df = 15; p = 0.61). These results were not related to the effects of LPS (Fig. 8).
4. Discussion The pigmentation on the surface of the testes increased rapidly (approximately 2 h after inoculation) in response to the exposure to LPS and decreased to control levels 24 h after systemic administration of LPS. This immediate increase was also evident on the kidneys, where we observed an increase of pigmentation 6 h after LPS exposure. In contrast, the pigmentation on the heart increased only after 12 h. However, the pigmentation did not vary in other cardiorespiratory organs. No changes related to LPS administration were observed in the organs of the digestive system or in the mesentery, the nerves of the lumbar plexus and the lumbosacral parietal peritoneum.
The function of the pigmentation in the visceral organs of frogs is not yet clear. Melanin has a protective role against free radicals and other potentially toxic agents (Zuasti et al., 1989; Agius and Roberts, 2003) and is a bactericidal agent (Christiansen et al., 1996). We observed an increase in the pigmentation of some organs after LPS exposure. Pigmentation increased on the testes. The protective role of melanin against the toxic effects of endotoxemia (i.e., oxygen burst and oxidative and nitrosative stress) may be involved in this response. Testicular dysfunction after contact with LPS has been reported in mammals: the germ cells undergo apoptosis and spermatogenesis is affected (Kajihara et al., 2006; Reddy et al., 2006). In the current study, 24 h after LPS administration, the pigmentation of the testes returned to control levels. Agius and Roberts (2003) reported an increase in pigmentation in the hematopoietic organs of fish related to pathology. This finding suggests that these cells and the pigmented cells present on the testes, the kidneys and the heart may have a similar functional role. Divya et al. (2010) reported a signaling action of melanocytes in the tail skeletal muscle of tadpoles at metamorphosis. This result suggests that melanin plays a role in the cascade of events leading to tail resorption in anuran larvae. Pigment cells on the testes are not described for most species, although some authors (e.g., Oliveira et al., 2002, 2003; Oliveira and Zieri, 2005; Zieri et al., 2007) have found a close relationship between visceral melanocytes and the vascular system as well as with the blood vessels and the associated connective membranes of
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Fig. 8. Comparative analysis of the pigmentation of the mesentery, parietal peritoneum and lumbar nerve plexus in Eupemphix nattereri inoculated with LPS from Escherichia coli. CONT, control group; LPS 2 h, 6 h, 12 h, 24 h and 48 h: animals analyzed 2, 6, 12, 24 or 48 h after LPS administration, respectively.
other organs. The pigmentation is present in the interstitium and tunica albuginea of the testes of E. nattereri, Physalaemus cuvieri and Physalaemus marmoratus and gives the testes a completely or partially black appearance (Oliveira et al., 2002, 2003; Oliveira and Zieri, 2005; Zieri et al., 2007). The association of pigmentation with the germinative locules suggests that pigmented cells may be protecting germ cells against free radicals. The visceral pigmentation of internal organs varies depending on intrinsic and extrinsic factors. The intrinsic factors include age and the nutritional and pathological status of the animal (Agius and Agbede, 1984). In addition, low temperatures have been shown to increase the amount of melanin and to induce metabolic and structural changes in hepatic cells of Rana esculenta (Barni et al., 1999).
The visceral pigmentation on the testes of Rhinella schneideri changes during the reproductive period. The pigmentation on the surface of these organs increases at the end of the reproductive season (Moresco and Oliveira, 2009). This observation is consistent with the action of melanin during spermatogenesis, a period with high testicular activity. Nevertheless, in other regions, such as the pericardium, the heart, the stomach, the intestine, the rectum, the nerves of the lumbar plexus, the lumbosacral parietal peritoneum and the mesentery, there were no changes in surface pigmentation related to the reproductive period (Moresco and Oliveira, 2009). However, the systemic administration of LPS promoted an immediate increase of pigmentation in the kidneys and the heart. This result demonstrates that visceral cells react against an actual product of bacteremia by producing melanin.
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The LPS from E. coli administered experimentally caused changes in the superficial pigmentation of the testes, kidney and heart. This observation suggests that this visceral pigmentation is a response to one or more products of LPS exposure. Therefore, these findings could be explained by the bactericidal role of melanin (Christiansen et al., 1996) because melanin protects cells and organs against products of LPS exposure. In the testes, the pigmented cells protect the germ cells against products of real bacteremia that could cause damage and influence the reproductive output directly, thus decreasing the fitness of the animals. Acknowledgements We thank Diogo B. Provete, M.Sc., Rafaela M. Moresco, M.Sc., and Dr. Amilcar S. Damazo for suggestions on earlier drafts of the manuscript, Dr. Rodrigo Zieri and Dr. Lia R. de Souza Santos for helping with field work and the Fundac¸ão de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for providing grants (05/02919-5 and 2009/13925-7) and awarding a master’s scholarship to FLB (Proc. 2008/52389-0). We also thank IBAMA for the collecting permits (RAN-IBAMA 18573-1). References Agius, C., 1980. Phylogenetic development of melano-macrophage centers in fish. J. Zool. 191, 11–31. Agius, C., Agbede, S.A., 1984. An electron microscopical study on the genesis of lipofuscin, melanin and haemosiderin in the haemopoietic tissues of fish. J. Fish Biol. 24, 471–488. Agius, C., Roberts, R.J., 2003. Review: melano-macrophage centres and their role in fish pathology. J. Fish Biol. 26, 499–509. Bagnara, J., Matsumoto, J., 2006. Comparative anatomy and physiology of pigment cells in non-mammalian tissues. In: Nordlund, J.J., Boissy, R.E., Hearing, V.J., King, R.A., Ortonne, J.P. (Eds.), The Pigmentary System: Physiology and Pathophysiology. Oxford University Press, New York, pp. 9–40. Barni, S., Bertone, V., Croce, A.C., Bottiroli, G., Bernini, F., Gerzeli, G., 1999. Increase in liver pigmentation during natural hibernation in some amphibians. J. Anat. 195, 19–25. Christiansen, J.L., Grzybowski, J.M., Kodama, R.M., 1996. Melanomacrophage aggregations and their age relationships in the yellow mud turtle, Kinosternon flavescens (Kinosternidae). Pigm. Cell Res. 9, 185–190. Divya, L., Beyo, R.S., Sreejith, P., Akbarsha, M.A., Oommen, V.O., 2010. Skeletal muscle–melanocyte association during tadpole tail resorption in a tropical frog, Clinotarsus curtipes Jerdon (Anura, Ranoidea). Zoology 113, 175–183. Fenoglio, C., Boncompagni, E., Fasola, M., Gandini, C., Comizzoli, S., Milanesi, G., Barni, S., 2005. Effects of environmental pollution on the liver parenchymal cells and
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